Targeting protein degradation pathways for Oncology drug discovery has recently been validated by the success of the proteasome inhibitor (bortezomib) in the treatment of blood cancers. However, bortezomib has side effects, and cancer patients can also develop resistance to the drug. We have developed a rationale and unique strategy to identify new drug candidates that act more selectively than the proteasome in protein degradation pathways. We will exploit a class of enzymes upstream in the pathway, known as E3 ubiquitin ligases, for drug inhibition-since each E3 acts on much fewer substrates than the proteasome, we expect less toxic drugs to be developed. Despite their great potential as drug targets, discovering and bringing E3 inhibitors to the clinic have remained unmet challenges. To achieve these goals, we have designed an innovative approach with the aim to identify inhibitors of Mdm2, the main E3 ligase for the tumor suppressor protein p53 --Mdm2 inhibition is known to cause an increase in p53 protein levels in cells and is expected to be pharmacologically relevant. The Mdm2 inhibitors we find will then undergo proof-of-concept studies for early-stage drug development. Specifically, following target-based biochemical screens for Mdm2 inhibitors, the most potent hits that exhibit desirable drug-like features and are selective over other E3 ligases will undergo initial medicinal chemistry efforts. Top-performing compound analogs will then be characterized for the ability to elicit the predicted biological and therapeutic effects. Finally, we will investigate their mechanism of E3 inhibition using biochemical and structural biology approaches, followed by a second round of medicinal chemistry and validation studies. Relevance: The Mdm2 oncoprotein has been widely validated as an oncology drug target. It is amplified or overexpressed in 7% of all human cancers, estimated to impact 100,000 new patients every year in the U.S. alone. However, no drugs targeting Mdm2 or other E3 ligases are currently in clinical trials or in the market. The discovery of inhibitors of Mdm2 E3 ligase activity with anti-tumor properties will thus represent a significant innovation in the development of drugs for the treatment of cancer.